Synthesis and 15N NMR characterization of 4-vinylbenzyl substituted bases of nucleic acids

Article abstract of DOI:10.1002/jhet.5570400418

The following substances have been synthesized and characterized as monomers or intermediates for syntheses of new polymers: 1-(4-vinylbenzyl) uracil (1a), 1-(4-vinylbenzyl)thymine (1b), N-4-acetyl-1-(4-vinylbenzyl) cytosine (1c), 1-(4-vinylbenzyl)cytosin

Full text of DOI:10.1002/jhet.5570400418

Synthesis and 15N NMR Characterization of
671
Jul-Aug 2003
4-Vinylbenzyl Substituted Bases of Nucleic Acids
ˇ
˚
Milosˇ Sedlák* [1], PetrSimunek [1] and Markus Antonietti [2]
[1] Department of Organic Chemistry, Faculty of Chemical Technology, University of Pardubice,
532 10 Czech Republic
[2] Max-Planck Institute of Colloids and Interfaces, Research Campus Golm,14424 Postdam,Germany
Received January 21, 2003
The following substances have been synthesized and characterized as monomers or intermediates for
syntheses of new polymers: 1-(4-vinylbenzyl)uracil (1a), 1-(4-vinylbenzyl)thymine (1b), N-4-acetyl-1-(4-
vinylbenzyl)cytosine (1c), 1-(4-vinylbenzyl)cytosine (1d), 9-(4-vinylbenzyl)adenine (2a), 2-amino-9-(4-
vinylbenzyl)-6-chloro-9H-purine (2b), and 9-(4-vinylbenzyl)-guanine (2c). The alkylation reactions with 4-
vinylbenzyl chloride were catalyzed with anhydrous sodium iodide. The substitution at position 9 in
substances 2a-c was confirmed by 15N NMR.
J. Heterocyclic Chem., 40, 671(2003).
Introduction.
ethyldisilazane (HMDS) using trimethylsilyl chloride
(TMSC) as the catalyst. However, in our case the alkyla-
tion was catalysed with anhydrous NaI with addition of
hydroquinone as polymerization inhibitor (Scheme 1).
After the alkylation, the silyl groups were removed by
hydrolysis: the yields of monomers 1a and 1b after crystal-
lization were 55 and 48%, respectively.
1-(4-Vinylbenzyl)cytosine (1d) was prepared in analo-
gous way from N-4-acetylcytosine: after the silylation,
alkylation and hydrolysis we obtained first N-4-acetyl-1-
(4-vinylbenzyl)cytosine (1c), which was subsequently
submitted to methanolysis to give 1d (Scheme 1). The
yields of monomers 1c and 1d after crystallization were 50
and 77%, respectively.
Purine and pyrimidine bases represent a significant part
of nucleic acids, which substantially contribute to the
mechanism of storage and transfer of genetic information.
The present era is characterized by an intensive develop-
ment of molecular biology, which necessitates a detailed
study of mechanisms at the level of nucleic acids [1]. The
template polymerization plays a key role in synthesis of
biopolymers, such as DNA, RNA and proteins [1]. In the
case of nucleic acids, the template polymerization is char-
acterized by an extremely precise arrangement of the indi-
vidual bases in biopolymer in accordance with the daugh-
ter template, with respect to both the sequence and the
chain length [1]. On the other hand, synthetic polymers are
usually much less defined from the standpoint of both the
control over molecular mass distribution and the polymer-
ization degree. In earlier work [2,3], some monomers for
radical template polymerization were prepared on the basis
of esters of methacrylic and acrylic acids corresponding to
uracil or adenine derivatives, and these were used for
preparation of complementary polymers.
Scheme 1
The aim of the present work was to synthesize and charac-
terize monomers derived from purine and pyrimidine bases.
For the monomer units we chose pyrimidine bases substi-
tuted at 1-position and purine bases substituted at 9-position
with 4-vinylbenzyl substituent. These monomers will be
used to study the possibilities of the template polymerization
itself and, moreover, they can also be applied to preparation
of a number of modified polymers with unique properties.
Another example of application can consist in a modified
packing of a chromatographic column that can be used for
separation of nucleosides and related compounds [4,5].
Results and Discussion.
1. Discussion of Synthesis.
The synthesis of 1-(4-vinylbenzyl)uracil (1a) and 1-(4-
vinylbenzyl)thymine (1b) was realized by the method [6]
of alkylation of the silylated bases. The silylation of uracil
and thymine was achieved by 24-h refluxing with hexam-
(a) HMDS/TMSC; reflux 24 hrs., (b) 4-vinylbenzylchloride, Nal/DMF
80°C 8-16 hrs., (c) H O, r.t./10 min., (d) NH /CH OH r.t. overnight.
2
3
3

ˇ
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672
M. Sedlák, PetrSimunek and M. Antonietti
Vol. 40
9-(4-Vinylbenzyl)adenine (2a) was prepared by analogous
spectroscopy [9]. The study of protons and carbon atoms
provides an only indirect piece of information about the
nitrogen skeleton, which is most sensitive to the substitu-
tion type. Therefore, very useful for this purpose
method of alkylation of adenine with allyl chloride [7] or
benzyl chloride [8]. However, in this case the alkylation of
adenine with 4-vinylbenzyl chloride was catalyzed by anhy-
drous NaI. The synthesis was carried out by boiling in DMF
under reflux, in the presence of K CO and hydroquinone
15
appeared to be the techniques based on N NMR spec-
15
troscopy [10-12]. Thorough N NMR studies of selected
2
3
7
9
for 16 hrs (Scheme 2). The minor product – 7-(4-vinylben-
zyl)adenine – was not isolated and was separated on silica
gel and by crystallization; the overall yield of 2a was 35%.
9-(4-Vinylbenzyl)guanine (2c) was synthesized by alky-
lation of 2-amino-6-chloro-9H-purine in boiling DMF
N /N -substituted purine analogues carried out by
Remaud et al.[11] showed substantial differences in elec-
7
9
tron structures of the N /N isomers. This paper [11] and
the results published by Marek et al.[12] resulted in cer-
tain conclusions that are useful in diagnosis of substitu-
tion type in purine derivatives. Perhaps the most useful is
under reflux with added K CO and hydroquinone for 32
2
3
hrs. 2-Amino-7-(4-vinylbenzyl)-6-chloro-9H-purine was
not isolated and was separated by flash chromatography
on silica gel. The crystallization of evaporated eluate first
gave 2-amino-9-(4-vinylbenzyl)-6-chloro-9H-purine (2b)
(33% yield), whereupon the chlorine at 6-position of
7
the chemical shift of N-3 nitrogen. In N this nitrogen
atom is less shielded by about 18-20 ppm as compared
9
with the N isomer.
15
The N NMR parameters of synthesized purine deriva-
tives 2a and 2c are presented in Table 1. Table 2 presents
purine 2b was substituted (S ) by hydroxyl ion, which
N
15
7
9
the N NMR parameters of selected N and N isomers
gave 9-(4-vinylbenzyl)guanine (2c) in the yield of 64%
after recrystallization.
1
15
for comparison. Figure 1 gives the H- N HMBC spec-
trum of compound 2c. A comparison of N chemical
15
shifts of compounds 2a with 3a, and 2c with 4a, shows a
good accordance between the individual derivatives.
Wherefrom it follows that the substances 2a,c prepared by
Scheme 2
9
us are N substituted purine derivatives.
2.2 Pyrimidine Derivatives.
The nitrogen chemical shifts of compounds 1a-d are
1
15
given in Table 1. The H- N gs HMBC spectrum of
thymine derivative 1b is presented in Figure 2. On the
whole, the results are comparable with the data obtained
on analogous nucleosides [13,14].
2. Discussion of NMR Results.
The crystal structures of 1-(4-vinylbenzyl)uracil (1a)
and 9-(4-vinylbenzyl)adenine (2a) appeared [15] during
completion of our work.
2.1. Purine Derivatives.
As the syntheses of N-alkylated purine derivatives usu-
7
9
ally produce mixtures of N and N regio-isomers, it is
important for one to be able to differentiate between these
regio-isomers. Application of multinuclear magnetic reso-
nance techniques proved useful for this purpose. Recently,
certain empirical rules have been developed, which are
based on differences in chemical shifts of certain diagnos-
EXPERIMENTAL
Measurement of NMR Spectra.
The 1H and13C NMR spectra were measured on a Bruker AMX
360 apparatus equipped with a 5 mm broadband probe and on a
Bruker Avance 500 apparatus equipped with a 5 mm broadband
1
13
tic carbon signals with application of 2D H- C NMR

Jul-Aug 2003
4-Vinylbenzyl Substituted Bases of Nucleic Acids
673
Table 1
N Chemical Shifts of 4-Vinylbenzyl Substituted Bases of Nucleic Acids
probe with Z-gradients and a with triple resonance probe with
Z-gradients (360.14 resp. 500.13 MHz for 1H and 90.57 resp
125.77 MHz for 13C). For the measurement, the substances were
dissolved in hexadeuteriodimethyl sulfoxide (DMSO-d6). The
15
Compound
N-1
N-3
N-7
N-9
NHR
1a
1b
1c
1d
2a
2c
-243.1
-246.6
-218.5
-233.8
–146.2
–176.0
–222.1
–224.8
-145.3
[a]
–156.6
–192.8
–231.5
–288.6
[a]
–140.7
–140.9
–219.1
–222.3
–299.3
[a] Not detected.
Table 2
15
7
9
N NMR Parameters of Selected N /N Isomers of
Substituted Adenines and Guanines
Compound
N-3
N-7
N-9
3a (ref. [11])
3b (ref. [11])
4a (ref. [12])
4b (ref. [12])
–156.8
–138.5
–198.9
–180.6
–142.2
–222.8
–132.5
–216.1
–216.2
–137.6
–225.1
–131.2
chemical shifts dH and dC are referenced to the middle signal of
multiplet of the solvent (dH = 2.55 ppm, dC = 39.6 ppm).
The assignment of individual signals was carried out on the
1
basis of H-H COSY, gs H-13C HSQC and gs 1H-13C HMBC
spectra. The carbon spectra were measured both in usual way and
by the APT pulse sequence.
1
15
Figure 1. H– N HMBC spectrum of 2c.
15N NMR spectra were measured at 36.50 and 50.69 MHz,
respectively. The spectra were measured by both direct detection
1
and by means of the gradient selected H-15N HMBC technique.
The standardization was carried out with the use of neat
nitromethane CH315NO2 (d = 0.0 ppm) placed in coaxial capillary.
Delays for long-range couplings were set to 10 ms and to 120 ms.
Syntheses of Substances.
1-(4-Vinylbenzyl)uracil (1a) and 1-(4-vinylbenzyl)thymine
(1b): A solution of uracil (2.25 g; 20 mmol) or thymine (2.50 g;
20 mmol) in hexamethyldisilazane (13 ml) was treated with
trimethylsilyl chloride (1 ml), and the reaction mixture was
refluxed under inert atmosphere (argon) for 24 hrs. The excess
hexamethyldisilazane was distilled off in vacuum. The raw sily-
lated uracil or thymine was mixed with a mixture of DMF (10
ml), 4-vinylbenzyl chloride (3 ml; 21 mmol), NaI (30 mg; 0.2
mmol) and hydroquinone (10 mg). The reaction mixture was
stirred at 80 °C for 8 hrs, whereupon it was poured in water (150
ml) and after 10-min stirring extracted with dichloromethane (2 ´
80 ml). The combined extracts were dried over MgSO4, filtered,
and concentrated in a vacuum evaporator. The evaporation
residue was recrystallized from methanol to give 2.5 g (55%)
white crystals of 1a, melting at 185-187 °C or 2.2 g (48%) white
crystals of 1b, melting at 162-165 °C. 1H NMR(1a): 4.90 (s, 2H,
H-7), 5.30 (dd, J 0.3 Hz, J 10.9 Hz, 1H, H-13b), 5.65 (dd, J 2.2
Hz, J 7.8 Hz, 1H, H-5), 5.86 (dd, J 0.3 Hz, J 17.7 Hz, 1H, H-13a),
6.76 (dd, J 17.7 Hz, J 10.9 Hz, 1H, H-12), 7.31-7.32 (m, 2H,
H-9), 7.50-7.51 (m, 2H, H-10), 7.80 (d, J 7.8 Hz, 1H, H-6), 11.39
(bs, 1H, H-3). 13C NMR (1a): 50.2 (C-7), 101.5 (C-5), 114.7 (C-
1
15
Figure 2. H– N HMBC spectrum of 1b.

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674
M. Sedlák, PetrSimunek and M. Antonietti
Vol. 40
13), 126.6 (C-10), 127.9 (C-9), 136.3 (C-12), 136.6 (C-11), 136.8
(C-8), 145.8 (C-6), 151.2 (C-2), 163.9 (C-4).
Anal. Calcd. for C13H12N2O2: ( 228.3 ): C, 68.41; H, 5.30; N,
12.27. Found: C, 68.28; H, 5.22; N, 12.18.
1H NMR (1b): 1.80 (d, J 1 Hz, 3H, CH3), 4.87 (s, 2H, H-7),
5.30 (d, J 10.9 Hz, 1H, H-13b), 5.86 (d, J 17.7 Hz, 1H, H-13a),
6.76 (dd, J 11 Hz, J 17.7 Hz, 1H, H-12), 7.31-7.33 (m, 2H, H-9),
.49 (m, 2H, H-10), 7.67 (d, J 1.2 Hz, 1H, H-6), 11.39 (bs, 1H,
H-3). 13C NMR (1b): 12.1 (CH3), 49.9 (C-7), 109.2 (C-5), 114.6
(C-13), 126.5 (C-10), 127.9 (C-9), 136.3 (C-12), 136.7 (C-8),
136.8 (C-11), 141.4 (C-6), 151.1 (C-2), 164.4 (C-4).
hot DMF (2 ´ 50 ml). The combined filtrates were evaporated in
vacuum until dry. The evaporation residue was mixed with chloro-
form (100 ml) and filtered with silica gel (30 g). The filtrate was con-
centrated to a volume of 30 ml and, while hot, treated with methanol
(30 ml). The crystals separated on cooling were collected by suction
and recrystallized from methanol with addition of charcoal. Yield
4.35 g (35%) 2a, m.p. 221-224 °C. 1H NMR (2a): 5.29 (d J 10.9 Hz,
1H, H-16b), 5.40 (s, 2H, H-10), 5.85 (d, J 17.7 Hz, H-16a), 6.74 (dd,
J 10.9 Hz, J 17.7 Hz, 1H, H-15), 7.30 (bs, 2H, NH2), 7.33-7.34 (m,
2H, H-12), 7.47-7.49 (m, 2H, H-13), 8.19 (s, 1H, H-2), 8.30 (s, 1H,
H-8). 13C NMR (2a): 46.0 (C-10), 114.7 (C-16), 118.8 (C-5), 126.5
(C-13), 128.0 (C-12), 136.2 (C-15), 136.7 (C-14), 136.8 (C-11),
140.9 (C-8), 149.6 (C-4), 152.8 (C-2), 156.1 (C-6).
Anal. Calcd. for C14H14N2O2: (242.3) C, 69.41; H, 5.82; N,
11.56. Found: C, 69.47; H, 5.33; N, 11.31.
Anal. Calcd. for C14H13N5 : ( 251.3): C, 66.92; H, 5.21; N,
27.87. Found: C, 67.11; H, 5.10; N, 28.05.
N-4-Acetyl-1-(4-vinylbenzyl)cytosine (1c).
A solution of N-4-acetylcytosine (3.06 g; 20 mmol) in hexam-
ethyldisilazane (13 ml) was treated with trimythylsilyl chloride
(1 ml), and the reaction mixture was refluxed under inert atmos-
phere (argon) for 24 hrs. The excess hexamethyldisilazane was
distilled of in vacuum. The raw O,N-silylated N-4-acetylcytosine
was mixed with a mixture of DMF (10 ml), 4-vinylbenzyl chloride
(3 ml; 21 mmol), NaI (30 mg; 0.2 mmol), and hydroquinone
(10 mg). The reaction mixture was stirred at 80 °C for 16 hrs,
whereupon it was poured in water (200 ml), stirred for another 10
min, and extracted with dichloromethane (4 ´ 100 ml). The com-
bined extracts were dried over MgSO4, filtered, and concentrated
in a vacuum evaporator. The evaporation residue was recrystal-
lized from methanol to give 2.7 g (50%) 1c melting at 114-116 °C.
1H NMR (1c): 2.06 (s, 3H, CH3), 4.96 (s, 2H, H-7), 5.22 (d, J 11
Hz, 1H H-13b), 5.78 (d, J 17.7 Hz, 1H H-13a), 6.68 (dd, J 10.9 Hz,
J 17.7 Hz, 1H H-12), 7.15 (d, J 7.2 Hz, 1H H-5), 7.24-7.26 (m, 2H,
H-10), 7.41-7.42 (m, 2H, H-9), 8.19 (d, J 7.3 Hz, 1H, H-6), 10.83
(bs, 1H, NH). 13C NMR(1c): 24.5 (CH3), 52.2 (C-7), 95.5 (C-5),
114.7 (C-13), 126.5 (C-10), 12.2 (C-9), 136.3 (C-12), 136.6 (C-8),
136.7 (C-11), 150.4 (C-6), 155.4 (C-2), 162.6 (C-4), 171.1 (C=O).
Anal. Calcd. for C15H15N3O2: ( 269.3): C, 69.90; H, 5.61; N,
15.60. Found: C, 69.81; H, 5.55; N, 15.48.
2-Amino-9-(4-vinylbenzyl)-6-chloro-9-H-purine (2b).
A suspension of 2-amino-7-(4-vinylbenzyl)-6-chloro-9H-
purine (4 g; 24 mmol), anhydrous potassium carbonate (3.6 g; 26
mmol), and hydroquinone (10 mg) in DMF (50 ml) was stirred
under inert atmosphere (argon) and treated with 4-vinylbenzyl
chloride (3.4 ml; 12 mmol). The mixture was refluxed with exclu-
sion of air moisture for 32 hrs, whereupon it was hot filtered. The
filtrate was evaporated to dryness in vacuum. The evaporation
residue was separated by flash chromatography on silica gel
(100 g) using a CHCl3-CH3OH (20:1) mixture as eluent. The first
fraction was evaporated until dry, and the residue was recrystal-
lized from a benzene-CHCl3 (1:1) mixture to give 2.25 g (33%)
2b melting at 193-196 °C. 1H NMR (2b): 5.29 (d, J 11 Hz, 1H, H-
16b), 5.33 (s, 2H, H-10), 5.85 (d, J 17.6 Hz, 1H, H-16a), 6.74 (dd,
J 17.7 Hz, J 10.9 Hz, 1H, H-15), 7.00 (bs, 2H, NH2), 7.28-7.29 (m,
2H, H-12), 7.48-7.50 (m, 2H, H-13), 8.29 (s, 1H, H-8).
Anal. Calcd. for C14H12ClN5 : ( 285.7): C, 58.85; H, 4.23; N,
24.51; Cl, 12.41. Found: C, 58.98; H, 4.37; N, 24.66; Cl, 12.22.
9-(4-Vinylbenzyl)guanine (2c).
A suspension of 2b (2 g; 7 mmol) in 1 M NaOH (50 ml) was
stirred at room temperature overnight. After neutralisation with
HCl (1:1) (pH ~ 7), the raw product was collected by suction,
dried, and recrystallized from methanol to give 1.2 g (64%) 2c
1-(4-Vinylbenzyl)cytosine (1d).
A methanolic solution of 1c (1 g; 4 mmol) in 1.8 M NH3 (50
ml) was stirred at room temperature overnight. The mixture was
evaporated on a water bath until dry, the evaporation residue was
washed with water (30 ml) and recrystallized from methanol to
give 700 mg (77%) 1d as white crystals melting at 287-290 °C.
1H NMR (1d): 4.88 (, 2H, H-7), 5.29 (d, J 11 Hz, 1H, H-13b),
5.72 (d, J 7.2 Hz, 1H, H-5), 5.85 (d, J 17.7 Hz, 1H, H-13a), 6.75
(dd, J 10.9 Hz, J 17.7 Hz, 1H, H-12), 7.10 (b, 2H, NH2), 7.27-
7.29 (m, 2H, H-9), 7.47-7.49 (m, 2H, H-10), 7.72 (d, J 7.2 Hz,
1H, H-6). 13C NMR (1d): 51.1 (C-7), 93.8 (C-5), 114.4 (C-13),
126.3 (C-10), 127.9 (C-9), 136.4 (C-12), 136.4 (C-11), 137.8
(C-8), 14.1 (C-6), 155.9 (C-4), 166.1 (C-2).
1
melting at 186-189 °C. H NMR (2c): 5.27-5.30 (m, 3H, H-10,
H-16b), 5.84 (d, J 17.7 Hz, 1H, H-16a), 6.49 (bs, 2H, NH2), 6.73
(dd, J 10.9 Hz, J 17.7 Hz, 1H, H-15), 7.23-7.25 (m, 2H, H-12),
7.46-7.48 (2H, H-13), 7.99 (s, 1H, H-8). 13C NMR (2c): 45.7
(C-10), 113.8 (C-5), 114.6 (C-16), 126.5 (C-13), 127.5 (C-12),
136.2 (C-15), 136.6 (C-14), 137.0 (C-11), 139.9 (C-8), 154.2
(C-4), 160.1 (C-2), 160.8 (C-6).
Anal. Calcd. for C14H13N5O: (267.3 ): C, 62.91; H, 4.90; N,
26.20 Found: C, 63.11; H, 4.98; N, 26.11.
Acknowledgments.
Anal. Calcd. for C13H13N3O: ( 227.3): C, 68.71; H, 5.77; N,
18.49. Found: C, 68.82; H, 5.82; N, 18.37.
The authors wish to acknowledge the financial support
provided by the Max-Planck Society Germany and Ministry of
Education, Youth and Sports of the Czech Republic (Project CI
MSM 253 100 001).
9-(4-Vinylbenzyl)adenine (2a).
A suspension of adenine (6.75 g; 50 mmol), anhydrous potassium
carbonate (7.60 g; 55 mmol), NaI (90 mg; 0.6 mmol), and hydro-
quinone (10 mg) in DMF (100 ml) was stirred under inert atmos-
phere (argon) and treated with 4-vinylbenzyl chloride (7.1 ml; 50
mmol). The mixture was refluxed with exclusion of air moisture for
16 hrs, whereupon it was hot filtered, and the solid was washed with
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